Use of gram negative species to treat atopic dermatitis

ABSTRACT

Pharmaceutical compositions are disclosed that includes a therapeutically effective amount of a purified viable Gram negative bacteria and a pharmaceutically acceptable carrier. The pharmaceutical compositions are formulated for topical administration. Methods of treating atopic dermatitis using these pharmaceutical compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/184,498, filed Nov. 8, 2018, which is a continuation of U.S.application Ser. No. 15/939,066, filed Mar. 28, 2018, now U.S. Pat. No.10,195,236, which is continuation of PCT International No.PCT/US2017/028133, filed Apr. 18, 2017, which claims the benefit of U.S.Provisional Application No. 62/324,762, filed Apr. 19, 2016, each ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of dermatology, specifically to theuse of the topical application of viable Gram negative bacterial totreat atopic dermatitis.

BACKGROUND

The term “eczema,” often used to describe atopic dermatitis, was coinedin ancient Greece, and roughly translates to “to boil out.” Modernscience, however, recognizes the contribution of both host andenvironmental factors to this disease. Hallmarks of the disease includereduced barrier function, reduced innate immune activation, andsusceptibility to infections with Staphylococcus aureus. Predisposinghost factors are suggested by monogenic mutations in STAT3, filaggrin,and other genes associated with AD-like phenotypes (Lyons et al.,Immunology and allergy clinics of North America 35, 161-183 (2015);published online Epub February (10.1016/j.iac.2014.09.008)). Hostgenetic influences can be therapeutically modulated through topicalsteroids or calcineurin inhibitors (Boguniewicz and Leung, J AllergyClin Immunol 132, 511-512 e515 (2013); published online (Epub) August(10.1016/j.jaci.2013.06.030)). S. aureus contributes to AD pathogenesis,and can be mitigated by antibiotics (Boguniewicz and Leung, supra;Kobayashi et al., Immunity 42, 756-766). Recent work has revealed thatthe skin microbiome is significantly different between healthy controlsand patients with AD and that symptoms are associated with a loss ofcommensal diversity (Kong et al., Genome research 22, 850-859 (2012);published online (Epub) May (10.1101/gr.131029.111)). A need remains formethods for therapeutically targeting this dysbiosis and treating atopicdermatitis.

SUMMARY OF THE DISCLOSURE

It is disclosed herein that culturable Gram-negative bacteria (CGN) fromthe skin of healthy controls were associated with activation of innateimmunity, enhanced barrier function, and control of S. aureus. TheseGram negative bacteria are of use for treating atopic dermatitis.

In some embodiments, pharmaceutical compositions are disclosed thatinclude a therapeutically effective amount of a purified viable Gramnegative bacteria and a pharmaceutically acceptable carrier, wherein a)a lysate and/or component of the Gram negative bacteria inhibits growthof S. aureus in an in vitro assay; b) the Gram negative bacteriastimulates human keratinocytes; c) the Gram negative bacteria inducescytokine expression from human cells; and d) the Gram negative bacteriais non-pathogenic when administered to the skin of the subject. Thepharmaceutical compositions are formulated for topical administration.

Methods of treating a topical dermatitis using these pharmaceuticalcompositions are also disclosed.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D: CGN isolates differ in presence and S. aureus inhibitionbetween healthy volunteers and patients with AD. (A) Percentage ofindividuals with CGN isolate yield from HV (n=26) and AD (n=17)subjects. Individuals with multiple CGN isolates were counted perisolate for >100% total in HV; see Table S1 for details. (B) Eightstrains of S. aureus isolated from HV and AD patients were grown in thepresence of either CGN supernatant or control media. Each data pointrepresents the effect on S. aureus growth of supernatant from one CGNisolate compared to media control (HV isolates=9, AD isolates=7); shapesrepresent sourcing of S. aureus as either the participant's autologousS. aureus, or one S. aureus isolate from HV or AD patients. Data pointswith red outline represent one HV- and one AD-derived CGN isolateselected for subsequent human challenge and mouse model experiments.Data is alternatively presented by CGN species in Supplemental FIG. 1.(C) Healthy mouse ears were co-inoculated with S. aureus (strain SAAS9)and R. mucosa (Rm) from HV or AD patients, or P. aeruginosa from a HVfor 10 days. Day 12 ears were homogenized and plated by serial dilutionfor CFU. Percent change in growth versus diluent (no CGN added) controlis shown. (D) CGN CFU yield taken from same mice as panel C. Data shownare a combination of three or more independent experiments (B) orrepresentative of two independent experiments (C-D) and displayed asmean+sem. SA=S. aureus, HV=Healthy Volunteer, AD=Atopic Dermatitis,CGN=Culturable Gram-negative, Rm=Roseomonas mucosa, Pa=Pseudomonasaeruginosa. Significance determined by Student's t test (B) or ANOVAwith Bonferroni's correction (C-D).

FIGS. 2A-2D: CGN from HV enhances innate immunity and barrier function.(A) Cytokine analysis for in vivo human blister challenge on healthycontrols showing cytokine responses to a representative strain of R.mucosa from a healthy control versus one isolated from a patient with ADas compared to the saline control well (dotted line), N=7. (B) Pairedanalysis of data presented in panel A showing the cytokine production inthe blister well exposed to the HV sourced R. mucosa less the cytokineproduction in the blister well exposed to the AD sourced R. mucosa inthe same human subject. (C) Mouse ears were inoculated daily for threedays with 1e7 CFU R. mucosa from either HV or AD. Day 5 mRNA abundancefor IL-10 and filaggrin standardized to GAPDH and compared to untreatedmice is shown, N=4-5 mice per group. (D) Mouse backs were shaved andhair chemically removed on day 0. TEWL was then measured after dailyapplication of 1e7 CFU R. mucosa from either HV or AD, N=4-5 mice pergroup. Data shown are a combination of 7 independent experiments (A-B)or representative of two or more independent experiments (C-D) anddisplayed as mean+sem (C-D) or mean and individual participants (A-B).HV=Healthy Volunteer, AD=Atopic Dermatitis, CGN=CulturableGram-negative, Rm=Roseomonas mucosa, FLG=filaggrin, IL-=Interleukin,TEWL=Transepidermal Water Loss. Significance from diluent control (or asindicated) determined by ANOVA with Bonferroni's correction.

FIGS. 3A-3D: CGN from HV improves outcomes in the MC903 mouse model ofAD-like dermatitis. (A-B) Both ears of mice were inoculated daily withR. mucosa (Rm) from a HV or an AD patient, or P. aeruginosa (Pa) from aHV, or SAAS9 strain of S. aureus for 2 days prior to MC903 application.Then bacteria were co-applied with MC903 daily for 13 days. Day 14 earthickness (A) for each ear and serum total IgE levels (B) are shown;N=4-8 mice per group. (C-D) Mice were treated with MC903 for 14 days toinduce AD-like dermatitis. Starting on day 13, mice were treated dailyfor 3 day with 1e6 CFU of live R. mucosa from a HV or AD patient (HVCGNand ADCGN), 1e6 CFU of killed R. mucosa from the same HV re-suspended inthe supernatant from 3e6 CFU of autologous R. mucosa (Dead mix), orsupernatant from 1e7 CFU of the HV R. mucosa (Sup). Day 21 visualredness (D) and ear thickness (C) are shown; N=3-5 mice per group. Datashown are representative of three independent experiments and displayedas mean+sem. Significance determined by ANOVA.

FIG. 4: Species analysis of data from FIG. 1B. Eight strains of S.aureus isolated from HV and AD patients were grown in the presence ofeither CGN supernatant or control media. Each data point represents theeffect on S. aureus growth of supernatant from one CGN isolate comparedto media control (HV isolates=9, AD isolates=7). Significance between R.mucosa isolates determined by Student t-test.

FIGS. 5A-5C: Suction Blister Protocol. (A) Image of 3D printed blisterinduction device. (B) Blister results 2 hours after suction. (C)Challenge chamber placed over denuded blister areas, bacterial isolatesplaced via pipette into the center of each challenge cap.

FIGS. 6A-6B: CGN impacts on cytokine and antimicrobial peptideresponses. Cytokine analysis (A) and antimicrobial peptides (B) for invivo human blister challenge (see supplemental methods), N=5. Data shownare a combination of five independent experiments and displayed asmean+sem (B) or mean and individual participants (A).

FIGS. 7A-7G: CGN stimulate primary human keratinocytes. Primary humanforeskin keratinocytes were cultured to confluence. 1e7 CFU ofGram-negative bacteria were added per well. mRNA was harvested from theKC 24 hours later and analyzed by PCR Data is representative of threeindependent experiments and displayed as mean+sem with individual dotsrepresenting KC cultured with distinct isolates. Significance determinedby Student t-test. *=p<0.05, **=p>0.01.

FIGS. 8A-8C. Lipids produced by HV-R. mucosa inhibit S. aureus growth.(A) Ammonium sulfate precipitation was performed on supernatants from R.mucosa and P. aeruginosa prior to evaluating the S. aureus (strainUSA300) inhibition as performed in FIG. 1B. (B) Three isolates of S.aureus were cultured in the presence or absence oflysophosphatidylcholine (LPC) or cardiolipin with inhibition assessed asin FIG. 1B versus diluent (0). (C) Human foreskin keratinocytes werecultured in the presence or absence of LPC were assessed as in FIG. S3.Data is representative of three independent experiments and displayed asmean±sem. Significance determined by ANOVA with Bonferroni's correction.*=p<0.05.

FIG. 9: CGN impact mouse fildaggrin responses during MC903 challenge.Mice underwent MC903 treatment along with inoculation of Gram-negativeisolates as shown. mRNA was harvested from ears on day 14 and analyzedby PCR. Data shown are representative of three independent experimentsand displayed as mean+sem. Significant difference from MC903 shown ascalculated by ANOVA. *=p<0.05.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

It is disclosed herein that commensal organisms from healthy controlsdiffer in their immune activation, barrier function, and antimicrobialprofiles when compared to identical species taken from patients withatopic dermatitis. Thus, a live-biotherapeutic approach to treatingpatients with atopic dermatitis is provided.

Pharmaceutical compositions, formulated for topical administration, aredisclosed herein which can be used for the treatment of atopicdermatitis. These pharmaceutical compositions include a therapeuticallyeffective amount of a purified viable Gram negative bacteria and apharmaceutically acceptable carrier, wherein a) a lysate and/orcomponent of the Gram negative bacteria inhibits growth of S. aureus inan in vitro assay; b) the Gram negative bacteria stimulates humankeratinocytes; c) the Gram negative bacteria induces cytokine expressionfrom human cells; and d) the Gram negative bacteria is non-pathogenicwhen administered to the skin of the subject. In a specific non-limitingexample, the Gram negative bacteria produce lysophosphatidylcholine.

The Gram negative bacteria can be from any species. Thus, in certainexamples, if the Gram negative bacteria is Pseudomonas, the Gramnegative bacterium can be a Pseudomonas aeruginosa, Pseudomonas luteola,or Pseudomonas oryzihabitans. In other examples, if the Gram negativebacterium is Pantoea, the Gram negative bacterium can be Pantoeaseptica. In additional examples, if the Gram negative bacterium isMoraxella, the Gram negative bacterium can be Moraxella osloensis. Infurther examples the Gram negative bacterium is Roseomonas, the Gramnegative bacterium can be Roseomonas mucosa. The Gram negative bacteriaincluded in the pharmaceutical composition can be from a single strain,a single species, or a single Genus. Alternatively, combinations ofstrains, species and/or genera of Gram negative bacteria can be used inthe disclosed methods.

Terms

Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Atopic dermatitis: A chronic disease that affects the skin. In atopicdermatitis, the skin becomes extremely itchy. Scratching leads toredness, swelling, cracking, “weeping” clear fluid, and finally,crusting and scaling. In most cases, there are periods of exacerbationsfollowed by periods of remissions. Although it is difficult to identifyexactly how many people are affected by atopic dermatitis, an estimated20% of infants and young children experience symptoms of the disease.Approximately 60% of these infants continue to have one or more symptomsof atopic dermatitis in adulthood. Thus, more than 15 million people inthe United States have symptoms of the disease. The “lesion area” is theregion of the skin affected by atopic dermatitis. Generally a lesion ischaracterized by skin dryness (xerosis), redness, blisters, scabs, orany combination. A non-lesion area is not affected by atopic dermatitisor any other skin pathology.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibiotic: A compound or substance that kills or substantially slowsdown the growth of bacteria, fungus or any other microbe. An“antibacterial” is a compound or substance that kills or substantiallyslows the growth of bacteria.

Antibacterial antibiotics are commonly classified based on theirmechanism of action, chemical structure, or spectrum of activity. Mosttarget bacterial functions or growth processes. Those that target thebacterial cell wall (for example, penicillins and cephalosporins) or thecell membrane (for example, polymyxins), or interfere with essentialbacterial enzymes (for example, quinolones and sulfonamides) arebactericidal. Those that target protein synthesis (for example,aminoglycosides, macrolides, and tetracyclines) are generallybacteriostatic. Further categorization is based on their targetspecificity.

“Narrow-spectrum” antibacterial antibiotics target specific types ofbacteria, such as Gram-negative or Gram-positive bacteria.“Broad-spectrum antibiotics” affect a number of different types ofbacteria. Antibacterial agents also include cyclic lipopeptides (such asdaptomycin), glycylcyclines (such as tigecycline), and oxazolidinones(such as linezolid).

Topical antibiotics are antibiotics that are applied to a body surface,such as the skin or eye. Topical antibiotics are often formulated in anointment or a cream, and contain active agents such as macrolideantibiotic (such as erythromycin), a sulfa antibiotic (such assulfacetamide), a cyclic peptide (such as bacitracin a polymyxin) apsuedomonic acid (such as mupirocin), an ammyroglycoside (such asneomycin), or a quinolone (such as ciprofloxacin or ofloxacin), anitroimidazole (such as metronidazloe), or a combination of drugs (suchas bacitracin/polymyxin or neomycin/polymyxin B/bacitracin).

Commensal: Organisms are commensal when they can live in the sameenvironment and one benefits from another without affecting (eitherharming or benefitting) the other. Bacteria in the skin are consideredto be commensal with the host, such as a human. The number of commensalbacterial species present on skin bacteria can be detected, for example,using 16S ribosomal RNA to identify bacterial species present.

Epithelial Cell: A closely packed cell that forms an epithelium, such asin the skin. There are several types of epithelium, including simplesquamous epithelium, simple cuboidal epithelium, simple columnarepithelium, pseudostratified columnar epithelium, stratified squamous(nonkeratinized) epithelium, stratified cuboidal epithelium, andtransitional epithelium.

Gram-Negative Bacteria: Those bacteria having a plurality of exteriormembranes, an inner cell membrane, a thin peptidoglycan layer, and anouter membrane containing lipopolysaccharides (LPS). Porins exist in theouter membrane. Between the outer membrane and the cytoplasmic memberthere is space filled with periplasm. A S-layer is directly attached tothe outer membrane. Teichoic acids or lipoteichoic acids are notpresent. Most Gram negative bacteria, (but not all) do not form spores.Typical gram-negative species include but are not limited to those mostcommonly associated with sepsis and septic shock in humans, e.g., asreported in the HANDBOOK OF ENDOTOXINS, 1: 187-214, eds. R. Proctor andE. Rietschel, Elsevier, Amsterdam (1984). A conserved signature indel(CSI) in the HSP60 (GroEL) protein distinguishes all traditional phylaof gram-negative bacteria (e.g., Proteobacteria, Aquificae, Chlamydiae,Bacteroidetes, Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia,Planctomycetes, Spirochetes, and Acidobacteria). Gram negative bacterialinclude, but are not limited to, Escherichia coli, Klebsiellapneumoniae, Proteus species, Pseudomonas, Salmonella, and Roseomonasspecies. CGN are the gram negative bacterial found in the skin. By“component” is meant any molecule present in, or secreted by, the Gramnegative bacteria.

Thus, a component can be present in a lysate or a supernatant. Inspecific non-limiting examples, a component of a Gram negative bacteria,such as included in a supernatant, inhibits growth of S. aureus in an invitro assay.

Gram-Positive Bacteria: Bacteria characterized by a preponderance ofpeptidoglycans relative to LPS molecules in their membranes, which arecapable of inducing disease etiology and symptoms characteristic ofmicrobe infection, similar to those described for gram-negative species.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous is derived from a different cell ortissue type, or a different species from the recipient, and is clonedinto a cell that normally does not express that polypeptide.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Microbiome: The genetic content of the communities of microbes that livein and on the human body, both sustainably and transiently, includingeukaryotes, archaea, bacteria, and viruses (including bacterial viruses(i.e., phage)), wherein “genetic content” includes genomic DNA, RNA suchas micro RNA and ribosomal RNA, the epigenome, plasmids, and all othertypes of genetic information.

Isolated or Purified: An “isolated” or “purified” cell, such as Gramnegative bacteria, has been separated or purified away from other cellsor species in the environment which the cell, such as the bacteriaoccurs. The term “isolated” thus encompasses a bacterium purified bystandard purification methods, such as single cell cloning and culture.The term also embraces a bacterium prepared by recombinant methods orisolated from a natural source. Isolated (or purified) Gram negativebacteria are generally removed from other bacteria, such as Grampositive bacteria. Isolated Gram negative bacteria can be of a singlegenus, species, and/or strain. The term “substantially purified” as usedherein refers to a bacterial genus, species strain or a mixture of morethan one bacterial strains (e.g. Gram negative bacteria) that aresubstantially enriched in a sample, such that other types of bacteria(e.g., Gram positive bacteria) are depleted. The sample can besubstantially purified or enriched for the bacterial strain or mixtureof strains of interest such that the sample is at least about 70%, 80%,85%, 90%, 95%, 99% or greater of the desired bacterial genus species,strain(s) or less than about 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the undesirable or otherbacterial genus, species or strains.

Inhibiting or treating a disease: Inhibiting a disease, such as atopicdermatitis, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms or adecrease in lesion size. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease, such asredness, swelling, cracking, “weeping” clear fluid, and finally,crusting and scaling of the skin.

Interleukin (IL)-6. An IL that acts as both a pro-inflammatory cytokineand an anti-inflammatory myokine. IL-6 signals through a cell-surfacetype I cytokine receptor complex consisting of the ligand-binding IL-6Rachain (CD126), and the signal-transducing component gp130 (also calledCD130). CD130 is the common signal transducer for several cytokinesincluding leukemia inhibitory factor (LIF), ciliary neurotropic factor,oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitouslyexpressed in most tissues. In contrast, the expression of CD126 isrestricted to certain tissues. As IL-6 interacts with its receptor, ittriggers the gp130 and IL-6R proteins to form a complex, thus activatingthe receptor. Exemplary amino acid sequences are provided in Uniprotdatabase Accession No. P05231 (human) and P08505 (mouse) and exemplarymRNA sequences encoding IL-6 (along with the corresponding proteinsequence) are provided in GENBANK® Accession Nos. NM_000600.4 (human),Jan. 5, 2016, and NM_031168.2 (mouse), Oct. 26, 2015, which are allincorporated by reference herein.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Pharmaceutical agent: A bacteria, such as a Gram negative bacteria,chemical compound, nucleic acid molecule, or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. In one embodiment, a pharmaceutical agent isa Gram negative bacterium.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of a Gram negative bacteria hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate. Acceptable carriers also include creamsand ointments, such as for topical administration.

Pseudomonas: A genus of Gram-negative, aerobic gammaproteobacteria,belonging to the family Pseudomonadaceae containing 191 describedspecies. The members of the genus demonstrate a great deal of metabolicdiversity, and consequently are able to colonize a wide range of niches.Members of this genus can be determined using 16S rRNA analysis.Generally, members of the genus are rod shaped, aerobic, non-sporeforming bacteria that have one or more polar flagella, and exhibitpositive oxidase and catalase tests.

Roseomonas: A genus of aerobic, Gram negative, rod shaped bacteriumassigned to the phylum Proteobacteria and the family Acetobacteraceae.In specific non-limiting examples, bacteria can be determined to be inthe genus Roseomonas by evaluating the nucleic acid sequence of 16S rRNAfrom the bacteria. Roseomonas can appear as plump cocci, coccobacilli,or short rods, depending on the species. Most strains grow on MacConkeyagar, and growth occurs at 25° C., 30° C. and 35 OC, and temperatures inbetween. Most strains also grow at 42° C. A pale pink growth pigment isproduced, see BERGEY'S MANUAL® of Systemic Bacteriology, Volume Two, TheProteobacteria, Part 3, Springer Science & Business Media, July 25, 206,pages 88-89, available on-line from Google books.

Therapeutically effective dose: A dose sufficient to treat atopicdermatitis. In one embodiment, a therapeutically effective dose is anamount of a Gram negative bacteria sufficient to reduced lesion size.

Topical application: A topically applied agent is applied only in aspecific area, and not throughout the body. In particular examples thecomposition is applied to the skin, such as in a lesion. For example thepharmaceutical composition can be applied in a pharmaceuticalpreparation to a lesion.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram-negative andgram-positive bacterial cell. Exemplary vectors include those forexpression in Gram negative bacteria.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Gram Negative Bacteria

Provided are pharmaceutical compositions that include isolated orsubstantially purified Gram negative bacteria and combinations of Gramnegative bacteria from intact human skin, or propagated from such Gramnegative bacteria. These Gram negative bacteria have the capacity tomeaningfully provide functions of a healthy microbiota or catalyze anaugmentation to the resident microbiome when administered to a subjectwith atopic dermatitis. In particular, compositions are provided thattreat, prevent, delay or reduce the symptoms of atopic dermatitis.

These compositions can include Gram negative bacteria isolated from asubject that does not have atopic dermatitis, such as a healthy subjectwithout any pathological condition of the skin. In some embodiments, thesubject does not have any pathological condition. The Gram negativebacteria can be isolated from the skin of the subject directly, or canbe propagated in vitro using standard techniques for culturing bacteria.However, the Gram negative bacteria can be obtained from other sources,as discussed below. The Gram negative bacteria can be Proteobacteria,Spirochaetaceae, Enterobacteriales, Fusobacterium polymorphum, or aSelenomonadales. The Gram negative bacteria can be diplococci,coccobacilli, cocci or a bacilli.

In some embodiments, disclosed are compositions formulated for topicaladministration that include an isolated or substantially purified viableGram negative bacteria and a pharmaceutically acceptable carrier,wherein a) a lysate and/or component of the Gram negative bacteriainhibits growth of S. aureus in an in vitro assay; b) the Gram negativebacteria stimulates human keratinocytes; c) the Gram negative bacteriainduces cytokine expression from human cells; and d) the Gram negativebacteria is non-pathogenic when administered to the skin of the subject.By “component” is meant any molecule present in, or secreted by, theGram negative bacteria. In specific non-limiting examples, asupernatant, which includes molecules secreted by the Gram negativebacteria, inhibits growth of S. aureus in an in vitro assay.

Provided herein are specific genera, species, strains, and combinationsof strains or species, originally found within the human skin microbiotaof a subject without atopic dermatitis, such as a healthy subject.

In some embodiments, these species/strains are able to significantlyreduce the rate of skin pathogen replication within the in vitro assay.These species/strains are capable of providing a safe and effectivemeans by which to affect the growth, replication, and disease severityof such bacterial pathogens. In some embodiments, bacterial compositionsare provided with the ability to exclude pathogenic bacteria.

Exemplary bacterial compositions are demonstrated to reduce the growthrate of a specific pathogen, such as S. aureus. A Gram negative bacteriawith the capacity to durably reduce S. aureus in the skin can beidentified using a methodology for estimating an Ecological ControlFactor (ECF) for constituents within the human microbiota. The ECF isdetermined by assessing the antagonistic activity of a given commensalstrain or combination of strains towards a given pathogen (e.g., S.aureus) using an in vitro assay, resulting in observed levels ofecological control at various concentrations of the added commensalstrains. The ECF for a commensal strain or combination of strains issomewhat analogous to the longstanding minimal inhibitory concentration(MIC) assessment that is employed in the assessment of antibiotics. TheECF allows for the assessment and ranking of relative potencies ofcommensal strains and combinations of strains for their ability toantagonize skin pathogens. The ECF of a commensal strain or combinationof strains can be calculated by assessing the concentration of thatcomposition that is able to mediate a given percentage of inhibition(e.g., at least 10%, 20%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) ofa target pathogen (e.g., S. aureus) in the in vitro assay.

In some embodiments, the Gram negative bacteria stimulates humankeratinocytes. The Gram negative bacteria can stimulate keratinocytes invivo and/or in vitro. Gram negative bacteria stimulate keratinocytes byincreasing the transcription of the mRNA of immune mediators ormolecules involved in epithelial barrier function, such as production ofan mRNA encoding IL-1β, an mRNA encoding defensin beta 4, an mRNAencoding Cyp27b1, an mRNA encoding a vitamin D receptor, an mRNAencoding occludin, an mRNA encoding claudin 1, and/or an mRNA encodingfilaggrin.

In additional embodiments, the Gram negative bacteria induces cytokineexpression from human cells. Human cells include, but are not limitedto, the cells of the skin such as fibroblasts and keratinocytes.Cytokines include, but are not limited to, an interleukin (IL) such asIL-6 and IL-1β.

In yet other embodiments, the Gram negative bacteria producelysophosphatidylcholine.

In further embodiments, the Gram negative bacteria is non-pathogenicwhen administered to the skin of the subject. Generally, the Gramnegative bacteria does not cause any infection when administered tointact human skin. Thus, no pathogenesis is observed followingtreatment.

The viable Gram negative bacteria included in the disclosed compositioncan be of any genus. In some embodiments, the Gram negative bacteria isPseudomonas. In additional embodiments, the Gram negative bacteria isPantoea or a Moraxella. In other embodiments, the Gram negative bacteriais Roseomonas.

Bacteria from only a single genus can be included in the pharmaceuticalcomposition. Alternatively, combinations of genera can be included in apharmaceutical composition and are of use in the disclosed methods.Thus, the composition can include, for example, 1, 2, 3, 4, or 5 generaof Gram negative bacteria. In one specific non-limiting example, thecomposition includes viable Roseomonas. In another specific non-limitingexample, the compositions includes viable Pseudomonas. In yet anotherspecific non-limiting example, the compositions includes viableRoseomonas and viable Pseudomonas.

The viable Gram negative bacteria can be from any species. Thus, incertain examples, if the Gram negative bacteria is Pseudomonas, the Gramnegative bacterium can be a Pseudomonas aeruginosa, Pseudomonas luteola,or Pseudomonas oryzihabitans. In other examples, if the Gram negativebacterium is Pantoea, the Gram negative bacterium can be Pantoeaseptica. In additional examples, if the Gram negative bacterium isMoraxella, the Gram negative bacterium can be Moraxella osloensis. Infurther examples the Gram negative bacterium is Roseomonas, the Gramnegative bacterium can be Roseomonas aerilata, Roseomonas aerophila,Roseomonas aestuarii, Roseomonas alkaliterrae, Roseomonas aquatic,Roseomonas cervicalis, Roseomonas fauriae, Roseomonas frigidaquae,Roseomonas gilardii, Roseomonas lacus, Roseomonas ludipueritiae,Roseomonas mucosa, Roseomonas pecuniae, Roseomonas rhizosphaerae,Roseomonas riguiloci, Roseomonas rosea, Roseomonas soli, Roseomonasstagni, Roseomonas terrae, or Roseomonas vinacea. In one specificnon-limiting example, the Gram negative bacteria is Roseomonas mucosa.

Gram negative bacteria of a single species can be included in thepharmaceutical composition. Alternatively, combinations of species Gramnegative bacteria can be included in a pharmaceutical composition are ofuse in the disclosed methods. Thus, the composition can include 1, 2, 3,4, or 5 species of Gram negative bacteria. In one specific non-limitingexample, the composition includes viable Roseomonas mucosa. In anotherspecific non-limiting example, the compositions includes viablePseudomonas aeruginosa. In yet another specific non-limiting example,the compositions includes viable Roseomonas mucosa and viablePseudomonas aeruginosa.

The viable Gram negative bacterium of use can be from a single strain.Alternatively, the Gram negative bacteria can be from multiple strains.Gram negative bacteria of a single strain, or combinations of strainsGram negative bacteria can be included in the disclosed compositions areof use in the disclosed methods. Thus, the composition can include 1, 2,3, 4, or 5 species of Gram negative bacteria. In one specificnon-limiting example, the composition includes a single strain of viableRoseomonas mucosa. In a further specific non-limiting example, thecomposition includes 2, 3, 4, or 5 strains of viable Roseomonas mucosa.In another specific non-limiting example, the composition includes asingle strain of viable Pseudomonas aeruginosa. In a further specificnon-limiting example, the composition includes 2, 3, 4, or 5 strains ofviable Pseudomonas aeruginosa. In yet another specific non-limitingexample, the composition includes a strain of viable Roseomonas mucosaand a strain of viable Pseudomonas aeruginosa. In other specificnon-limiting examples, the composition includes 2, 3, 4 or 5 strains ofviable Roseomonas mucosa and 2, 3, 4 or 5 strains of viable Pseudomonasaeruginosa.

Thus, pharmaceutical compositions can include two types of Gram negativebacteria (“binary combinations” or “binary pairs”) or more than twotypes of Gram negative bacteria. In some embodiments, a pharmaceuticalcomposition can include at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, or at least 21, 22, 23,24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or atleast 40, at least 50 or greater than 50 types of Gram negativebacteria, as defined by genus, species and/or operational taxonomic unit(OTU), such as a strain. Generally the genus, species or strainindividually or in combination, have the following characteristics: a) alysate and/or component of the Gram negative bacteria inhibits growth ofS. aureus in an in vitro assay; b) the Gram negative bacteria stimulateshuman keratinocytes; c) the Gram negative bacteria induces cytokineexpression from human cells; and d) the Gram negative bacteria isnon-pathogenic when administered to the skin of the subject.

In certain embodiments, the Gram negative bacteria is transformed with aheterologous nucleic acid, such as in the form of a plasmid. Theexpression vector can encode any protein of interest. Exogenous DNA canbe introduced into bacterial cells with standard techniques such aselectroporation or calcium phosphate-mediated transfection.

In some embodiments, the heterologous nucleic acid is included in aplasmid. A plasmid generally contains multiple genetic elementspositionally and sequentially oriented with other necessary geneticelements such that the nucleic acid in a nucleic acid cassette can betranscribed and when necessary translated in the transfected cells.Plasmids include nucleic acids that are DNA derived from a plasmidvector, cosmids, or phagemids wherein one or more heterologous nucleicacid may be inserted. The heterologous nucleic acid can encode a proteinof interest, which can be operably linked to a promoter for expressionof the Gram negative bacteria.

Plasmids generally contain one or more unique restriction sites. Inaddition, a plasmid can confer some well-defined phenotype on the hostorganism which is either selectable or readily detected, such as drugresistance. Thus, the plasmid can include an expression cassette,wherein a polypeptide is encoded. Expression includes the efficienttranscription of an inserted gene, nucleic acid sequence, or nucleicacid cassette with the plasmid.

In one embodiment, when a circular plasmid is transferred into abacterial cell, it can be an autonomously replicating, extra-chromosomalDNA molecule, distinct from the normal bacterial genome and nonessentialfor bacterial cell survival under nonselective conditions. The term“persistent expression” as used herein refers to introduction of genesinto the cell together with genetic elements which enable episomal(extra-chromosomal) replication and/or maintenance of the geneticmaterial in the cell. This can lead to apparently stable transformationof the cell without the integration of the novel genetic material intothe chromosome of the host cell.

A plasmid can also introduce genetic material into chromosomes of thetargeted cell where it integrates and becomes a permanent component ofthe genetic material in that cell. Gene expression after stableintroduction can permanently alter the characteristics of the cell andits progeny arising by replication leading to stable transformation.

Methods for Preparing a Bacterial Composition for Administration

Methods for producing bacterial compositions may include three mainprocessing steps, combined with one or more mixing steps. The steps are:organism banking, organism production, and preservation.

For banking, the strains included in a bacterial composition may be (1)isolated directly from a specimen, such as, but not limited to, humanskin, or taken from a banked stock, (2) optionally cultured on anutrient agar or broth that supports growth to generate viable biomass,and (3) the biomass optionally preserved in multiple aliquots inlong-term storage.

The Gram negative bacteria can be isolated from the skin of a humansubject. Generally, the human subject does not have atopic dermatitis,or any other skin condition. Thus, the subject can be healthy, meaningthat they do not have any other pathological condition. However, Gramnegative bacteria can be isolated from other sources, such as commercialsources or environmental samples, and used in the methods andcompositions disclosed herein. As disclosed herein any Gram negativebacteria is of use, provided a) a lysate and/or component of the Gramnegative bacteria inhibits growth of S. aureus in an in vitro assay; b)the Gram negative bacteria stimulates human keratinocytes; c) the Gramnegative bacteria induces cytokine expression from human cells; and d)the Gram negative bacteria is non-pathogenic when administered to theskin of the subject. The Gram negative bacteria can then be propagated.

In embodiments using a culturing step, the agar or broth may containnutrients that provide essential elements and specific factors thatenable growth. A non-limiting example is a medium composed of 0.5 g/Ldextrose, 0.5 g/L yeast extract, 0.5 g/L proteose peptone, 0.5 g/Lcasamino acid, 0.3 g/L dipotassium phosphate, 50 mg/L magnesium sulfate,0.3 g/L sodium pyruvate. A variety of microbiological media andvariations are well known in the art (e.g. R. M. Atlas, Handbook ofMicrobiological Media (2010) CRC Press). Medium can be added to theculture at the start, may be added during the culture, or may beintermittently/continuously flowed through the culture. Thespecies/strains can be cultivated alone, or as an entire collectioncomprising the bacterial species/strains. As an example, a first strainmay be cultivated together with a second strain in a mixed continuousculture, at a dilution rate lower than the maximum growth rate of eithercell to prevent the culture from washing out of the cultivation.

The culture is incubated under favorable conditions for a timesufficient to build biomass. For bacterial compositions for human usethis is often at about 32-37° C., pH, and other parameter with valuessimilar to the normal human niche. The environment can be activelycontrolled.

When the culture has generated sufficient biomass, it may be preservedfor banking. The organisms may be placed into a chemical milieu thatprotects from freezing (such as by adding cryoprotectants), drying,and/or osmotic shock (such as by adding osmoprotectants), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then optionally treating the culture for preservation.Containers are generally impermeable and have closures that assureisolation from the environment. Cryopreservation can be accomplished byfreezing a liquid at ultra-low temperatures (e.g., at or below about−70° C.). Dried preservation removes water from the culture byevaporation (in the case of spray drying or cool drying) or bysublimation (e.g., for freeze drying, spray freeze drying). Removal ofwater improves long-term bacterial composition storage stability athigher temperatures. Strains and/or species can be cultured andpreserved individually, or species/strains can be mixed together forbanking.

In one non-limiting example, for cryopreservation, a bacterial culturecan be harvested by centrifugation to pellet the cells from the culturemedium, the supernate decanted and replaced with fresh culture brothcontaining 15% glycerol. The culture can then be aliquoted into 1 mLcryotubes, sealed, and placed at −80° C. or −70° C. for long-termviability retention. This procedure achieves acceptable viability uponrecovery from frozen storage.

Organism production may be conducted using similar steps to banking,including medium composition and culture conditions. Production can beconducted using large scale operation, especially for clinicaldevelopment or commercial production. At larger scales, there may beseveral subcultivations of the bacteria prior to the final cultivation.At the end of cultivation, the culture is harvested for formulation intoa dosage form for administration. This can involve concentration,removal of undesirable medium components, and/or introduction into achemical milieu that preserves the bacterial composition and renders itacceptable for administration via the chosen route. For one non-limitingexample, a bacterial composition may be cultivated to a concentration of10¹⁰ CFU/mL with a preservative medium consisting of 15% sucrose inwater.

Topical Formulations and Methods of Treatment

Pharmaceutical compositions are provided that include the disclosedisolated or substantially purified Gram negative bacteria, wherein thepharmaceutical composition is formulated for topical administration.These compositions include a pharmaceutically acceptable carrier, andoptionally include additional compounds. In some embodiments, thepharmaceutical composition includes additional active and/or inactivematerials in order to produce a final product, which may be in singledosage unit or in a multi-dose format.

Any subject that has atopic dermatitis can be treated using the methodsdisclosed herein. The subject can be a human. In some embodiments, thesubject is a child, such as a subject that is 11, 10, 9, 8, 7, 6, 5, 4,3, 2 or 1 year(s) of age or less. The subject can be an infant, such asa subject of less than 1 year of age. In other embodiments, the subjectis an adult, such as subject who is 18 years of age, greater than 20,25, 30, 35, 40, 45, 50, 55, or 60 years of age. The subject can be asenior, such as a subject who is greater than 65, 70, 75, or 80 years ofage. The subject can be immunocompromised or can have an intact immunesystem.

In some embodiments the pharmaceutical composition can include one ormore of a buffering agent, a preservative, a stabilizer, a binder, acompaction agent, a lubricant, a dispersion enhancer and/or a coloringagent. Non-limiting examples of suitable buffering agents include sodiumcitrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate,and calcium bicarbonate. Non-limiting examples of suitable preservativesinclude antioxidants, such as alpha-tocopherol and ascorbate, parabens,chlorobutanol, and phenol. Non-limiting examples of suitable bindersinclude sucrose, starches, pregelatinized starches, gelatin,polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides, andcombinations thereof. Non-limiting examples of suitable lubricantsinclude magnesium stearate, calcium stearate, zinc stearate,hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate,talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate,magnesium lauryl sulfate, and light mineral oil. A pH buffering agent(s)can, if employed and when dissolved in an aqueous component of thecomposition, provide a pH in the range of 5 to 7 (e.g. about pH 5.5).

The pharmaceutical composition can include other ingredients, such as tosustain growth of the bacteria. In some embodiments, the pharmaceuticalcomposition can include a nutrient. In some embodiments the compositioncomprises at least one carbohydrate. A “carbohydrate” refers to a sugaror polymer of sugars. The terms “saccharide,” “polysaccharide,”“carbohydrate,” and “oligosaccharide” may be used interchangeably. Mostcarbohydrates are aldehydes or ketones with many hydroxyl groups,usually one on each carbon atom of the molecule. Carbohydrates generallyhave the molecular formula C_(n)H_(2n)O_(n). A carbohydrate can be amonosaccharide, a disaccharide, trisaccharide, oligosaccharide, orpolysaccharide. The most basic carbohydrate is a monosaccharide, such asglucose, sucrose, galactose, mannose, ribose, arabinose, xylose, andfructose. Disaccharides are two joined monosaccharides. Exemplarydisaccharides include sucrose, maltose, cellobiose, and lactose.Typically, an oligosaccharide includes between three and sixmonosaccharide units (e.g., raffinose, stachyose), and polysaccharidesinclude six or more monosaccharide units. Exemplary polysaccharidesinclude starch, glycogen, and cellulose. Carbohydrates can containmodified saccharide units such as 2′-deoxyribose wherein a hydroxylgroup is removed, 2′-fluororibose wherein a hydroxyl group is replacewith a fluorine, or N-acetylglucosamine, a nitrogen-containing form ofglucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydratescan exist in many different forms, for example, conformers, cyclicforms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments the composition includes at least one lipid. A“lipid” includes fats, oils, triglycerides, cholesterol, phospholipids,fatty acids in any form including free fatty acids. Fats, oils and fattyacids can be saturated, unsaturated (cis or trans) or partiallyunsaturated (cis or trans). In some embodiments the lipid comprises atleast one fatty acid selected from lauric acid (12:0), myristic acid(14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid(17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid(18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoicacid (18:4), arachidic acid (20:0), eicosenoic acid (20:1),eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0).

In some embodiments the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In additional embodiments the composition comprises at least onesupplemental vitamin. The at least one vitamin can be fat-soluble orwater soluble vitamins. Suitable vitamins include but are not limited tovitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin,niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine,pantothenic acid, and biotin. Suitable forms of any of the foregoing aresalts of the vitamin, derivatives of the vitamin, compounds having thesame or similar activity of the vitamin, and metabolites of the vitamin.

Various other additives may be included in the compositions. Theseinclude, but are not limited to, antioxidants, astringents, perfumes,preservatives, emollients, pigments, dyes, humectants, propellants, andsunscreen agents, as well as other classes of materials whose presencemay be pharmaceutically or otherwise desirable. Non-limiting examples ofoptional additives are as follows: preservatives such as sorbate;solvents such as isopropanol and propylene glycol; astringents such asmenthol and ethanol; emollients such as polyalkylene methyl glucosides;humectants such as glycerine; emulsifiers such as glycerol stearate,PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate60; sorbitol and other polyhydroxyalcohols such as polyethylene glycol;sunscreen agents such as octyl methoxyl cinnamate (availablecommercially as Parsol MCX) and butyl methoxy benzoylmethane (availableunder the tradename Parsol 1789); antioxidants such as ascorbic acid(vitamin C), α-tocopherol (Vitamin E), β-tocopherol, γ-tocopherol,δ-tocopherol, ε-tocopherol, ζι-tocopherol, Z{circumflex over( )}-tocopherol, η-tocopherol, and retinol (vitamin A); essential oils,ceramides, essential fatty acids, mineral oils, vegetable oils (e.g.,soya bean oil, palm oil, liquid fraction of shea butter, sunflower oil),animal oils (e.g., perhydrosqualene), synthetic oils, silicone oils orwaxes (e.g., cyclomethicone and dimethicone), fluorinated oils(generally perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol),and waxes (e.g., beeswax, carnauba wax, and paraffin wax); skin-feelmodifiers; and thickeners and structurants such as swelling clays andcross-linked carboxypolyalkylenes.

Other additives include materials that condition the skin (particularly,the upper layers of the skin in the stratum corneum) and keep it soft byretarding the decrease of its water content and/or protect the skin.Such conditioners and moisturizing agents include, by way of example,pyrrolidine carboxylic acid and amino acids; organic antimicrobialagents such as 2,4,4′-trichloro-2-hydroxy diphenyl ether (triclosan) andbenzoic acid. Further additives include anti-inflammatory agents such asacetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeic agentssuch as retinoic acid; vasodilators such as nicotinic acid; inhibitorsof melanogenesis such as kojic acid; and mixtures thereof.

In other embodiments, the composition can include alpha hydroxyacids,alpha ketoacids, polymeric hydroxyacids, moisturizers, collagen, marineextract, and antioxidants such as ascorbic acid (vitamin C) and/ora-tocopherol (Vitamin E). Sunscreens may also be included. Additional,components such as enzymes, herbs, plant extracts, glandular or animalextracts can be added to the composition. The amounts of these variousadditives are those conventionally used in the cosmetics field, andrange, for example, from about 0.01% to about 20% of the total weight ofthe topical formulation.

The compositions can also include antimicrobial agents, to preventspoilage upon storage, i.e., to inhibit growth of microbes such asyeasts and molds. Suitable antimicrobial agents are typically selectedfrom the group consisting of the methyl and propyl esters ofp-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodiumbenzoate, sorbic acid, imidurea, and combinations thereof.

The compositions can also contain irritation-mitigating additives tominimize or eliminate the possibility of skin irritation or skin damageresulting from the chemical entity to be administered, or othercomponents of the composition. Suitable irritation-mitigating additivesinclude, for example: a-tocopherol; monoamine oxidase inhibitors,particularly phenyl alcohols such as 2-phenyl-1-ethanol; glycerin;salicylates; ascorbates; ionophores such as monensin; amphiphilicamines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine.The irritation-mitigating additive, if present, can be incorporated intothe compositions at a concentration effective to mitigate irritation orskin damage, typically representing not more than about 20 wt. %, moretypically not more than about 5 wt. %, of the formulation.

Further suitable pharmacologically active agents that may beincorporated into the present formulations in certain embodiments andthus topically applied along with the active agent include, but are notlimited to, the following: agents that improve or eradicate pigmented ornon-pigmented age spots, keratoses, and wrinkles; local anesthetics andanalgesics; corticosteroids; retinoids; and hormones. Some examples oftopical pharmacologically active agents include acyclovir,amphotericins, chlorhexidine, clotrimazole, ketoconazole, econazole,miconazole, metronidazole, minocycline, phenytoin, para-amino benzoicacid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone,dioxybenzone, tocopherol, tocopheryl acetate, zinc pyrithione,diphenhydramine, pramoxine, lidocaine, procaine, crotamiton,hydroquinone and its monomethyl and benzyl ethers, naproxen, ibuprofen,cromolyn, retinol, retinyl palmitate, retinyl acetate, coal tar,griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate,hydrocortisone 17-valerate, hydrocortisone 17-butyrate, progesterone,betamethasone valerate, betamethasone dipropionate, triamcinoloneacetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole,diphenylhydantoin, benzoyl peroxide, 5-fluorouracil, tacrolimus, andtopical steroids such as alclometasone, amcinonide, betamethasone,clobetasol, desonide, dexoximetasone, diflorasone, flucinonide,flurandrenolide, halobetasol, halcinonide, hydrocortisone, and/ortriamcinolone.

Although topical formulations, such as creams and salves formulated fordermal delivery are contemplated, the delivery systems can includetime-release, delayed release or sustained release delivery systems.Such systems can avoid repeated administrations of the compositions,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. Specific examples include, but are not limited to: (a)erosional systems such as those described in U.S. Pat. Nos. 4,452,775;4,667,014; 4,748,034; 5,239,660; and 6,218,371 and (b) diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480.

The delivery system can include collagen, fibrin, or a membrane extract,such as a basal membrane extract, for example wherein the composition isformulated for administration to the skin. Suitable basement membraneextracts include a biologically active polymerizable extract containingin parts by weight about 60-85% laminin, 5-30% collagen IV, 1-10%nidogen, 1-10% heparan sulfate proteoglycan and 1-5% entactin (see U.S.Pat. No. 4,829,000, incorporated herein by reference, which disclosesBME compositions as well as methods for producing these compositions).BME can support normal growth and differentiation of various cell typesincluding epithelial cells when cultured. Basal membrane extracts arewell known in the art and are commercially available.

For treatment of the skin, a therapeutically effective amount of thecomposition can be locally administered to the affected area. Thepharmacological compositions disclosed herein facilitate the use of atleast Gram negative bacterium for the treatment of atopic dermatitis.Such a composition can be suitable for delivery of the active ingredientto any suitable subject, such as but not limited to, a human subject,and can be manufactured in a manner that is itself known, e.g., by meansof conventional mixing, dissolving, granulating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmacologicalcompositions can be formulated in a conventional manner using one ormore pharmacologically (e.g., physiologically or pharmaceutically)acceptable carriers, as well as optional auxiliaries that facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically, as discussed above.

A composition can contain a single (unit) dose of Gram negativebacteria. Exemplary amounts are 10⁵-10¹² colony forming units (cfu) suchas 10⁶-10¹⁰, for example, 10⁵-10⁷ cfu, for example 10⁶ cfu. In someembodiments, suitable doses of Gram negative bacteria can be in therange 10⁴ to 10¹² cfu, e.g. one of 10⁴ to 10¹⁰, 10⁴ to 10⁸, 10⁶ to 10¹²,10⁶ to 10¹⁰, or 10⁶ to 10⁸ cfu. In other embodiments the composition caninclude at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%,about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% by weight ofbacteria. In other embodiments the composition can include at leastabout 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% toabout 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2%to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5%to about 5%, about 1% to 10 about 5%, by weight of the Gram negativebacteria.

The composition can be applied to the skin, such as at lesion areas andround lesion area, or at areas of intact skin (non-lesion areas) toprevent lesions for forming. The composition can be used to reducelesion size. The composition can be applied daily. The composition canbe applied 1, 2, 3, 4, or 5 time per day. The composition can be appliedevery other day, or 1, 2, 3, 4, 5, 6, or 7 times per week. Thecomposition can be applied weekly. In one specific, non-limitingexample, 10⁶ cfu is applied to the skin 2 or 3 times per week. Thecomposition can be formulated as a unit dose for administration.

Methods of producing topical pharmaceutical compositions such as creams,ointments, lotions, sprays and sterile aqueous solutions or suspensionsare well known in the art. Suitable methods of preparing topicalpharmaceutical compositions are described, for example in PCTPublication No. WO 95/10999, PCT Publication No. WO2012150269, U.S. Pat.No. 6,974,585, and PCT Publication No. WO 2006/048747, all incorporatedherein by reference. Optionally, a composition can include apharmaceutically acceptable viscosity enhancer and/or film former. Aviscosity enhancer increases the viscosity of the formulation so as toinhibit its spread beyond the site of application. Balsam Fir (Oregon)is an example of a pharmaceutically acceptable viscosity enhancer of usewith Gram negative bacteria.

A film former, when it dries, forms a protective film over the site ofapplication. The film inhibits removal of the active ingredient andkeeps it in contact with the site being treated. An example of a filmformer that is suitable for use in this invention is Flexible Collodion,USP. As described in Remington: The Science and Practice of Pharmacy,19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at page 1530,collodions are ethyl ether/ethanol solutions containing pyroxylin (anitrocellulose) that evaporate to leave a film of pyroxylin. A filmformer may act additionally as a carrier. Solutions that dry to form afilm are sometimes referred to as paints. Creams, as is well known inthe arts of pharmaceutical formulation, are viscous liquids or semisolidemulsions, either oil-in-water or water-in-oil.

Cream bases are water-washable, and contain an oil phase, an emulsifier,and an aqueous phase. The oil phase, also called the “internal” phase,is generally comprised of petrolatum and a fatty alcohol such as cetylor stearyl alcohol. The aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic or amphoteric surfactant.

Lotions, are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations in whichparticles, including the active agent, are present in a water or alcoholbase. Lotions are usually suspensions of solids, and preferably,comprise a liquid oily emulsion of the oil-in-water type. Lotions can beused for treating large body areas, because of the ease of applying amore fluid composition. It is generally necessary that the insolublematter in a lotion be finely divided.

Lotions typically contain suspending agents to produce betterdispersions as well as compounds useful for localizing and holding theactive agent in contact with the skin, e.g., methylcellulose, sodiumcarboxymethyl-cellulose, or the like.

Solutions are homogeneous mixtures prepared by dissolving one or morechemical substances (solutes) in a liquid such that the molecules of thedissolved substance are dispersed among those of the solvent. Thesolution may contain other pharmaceutically or cosmetically acceptablechemicals to buffer, stabilize or preserve the solute. Common examplesof solvents used in preparing topical solutions are ethanol, water,propylene glycol or any other acceptable vehicles.

Gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also, preferably,contain an alcohol, and, optionally, an oil. Some “organicmacromolecules,” of use, specifically gelling agents, are crosslinkedacrylic acid polymers such as the “carbomer” family of polymers, e.g.,carboxypolyalkylenes that re commercially available as CARBOPOL®. Alsoof use are hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol;cellulosic polymers such as hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and methyl cellulose; gums such as tragacanth and xanthangum, sodium alginate; and gelatin. In order to prepare a uniform gel,dispersing agents such as alcohol or glycerin can be added, or thegelling agent can be dispersed by trituration, mechanical mixing orstirring, or combinations thereof.

Ointments can also be used in the disclosed methods. Ointments aresemisolid preparations that are typically based on petrolatum or otherpetroleum derivatives. The specific ointment base to be used, as will beappreciated by those skilled in the art, is one that will provide for anumber of desirable characteristics, e.g., emolliency or the like. Anointment base is generally inert, stable, nonirritating, andnonsensitizing. Ointment bases are grouped in four classes: oleaginousbases; emulsifiable bases; emulsion bases; and water-soluble bases (seeRemington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.:Mack Publishing Co., 1995), at pages 1399-1404). Oleaginous ointmentbases include, for example, vegetable oils, fats obtained from animals,and semisolid hydrocarbons obtained from petroleum. Emulsifiableointment bases, also known as absorbent ointment bases, contain littleor no water and include, for example, hydroxystearin sulfate, anhydrouslanolin, and hydrophilic petrolatum. Emulsion ointment bases are eitherwater-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, andinclude, for example, acetyl alcohol, glyceryl monostearate, lanolin,and stearic acid. Water-soluble ointment bases are prepared frompolyethylene glycols of varying molecular weight.

Pastes are semisolid dosage forms in which the active agent is suspendedin a suitable base, and are also of use. Depending on the nature of thebase, pastes are divided between fatty pastes or those made fromsingle-phase aqueous gels. The base in a fatty paste is generallypetrolatum or hydrophilic petrolatum or the like. The pastes made fromsingle-phase aqueous gels generally incorporate carboxymethylcelluloseor the like as a base.

A topical composition can any form suitable for application to the bodysurface, such as a cream, lotion, sprays, solution, gel, ointment,paste, plaster, paint, bioadhesive, bandage, suspensions or the like,and/or may be prepared so as to contain liposomes, micelles, and/ormicrospheres. A topical composition can be used in combination with anocclusive overlayer so that moisture evaporating from the body surfaceis maintained within the formulation upon application to the bodysurface and thereafter.

A cream, lotion, gel, ointment, paste or the like may be spread on theaffected surface. A solution may be applied in the same way, but moretypically will be applied with a dropper, swab, sprayer or the like, andcarefully applied to the affected areas. The composition can be applieddirectly to the target location, for example in a topical preparationsuch as an ointment, or as a part of a dressing or a bandage. Thecomposition can be formulated as a unit dosage, for administration byany device for administration to the skin. The unit dosage may be areservoir of the active agent in a carrier, for example an adhesivecarrier capable of adhering to the skin for a desired period of timesuch as at least a day or more.

The pharmaceutical compositions are of use for the treatment of atopicdermatitis. Thus, in some embodiments, topical application results inreduced lesion size, reduce number of lesions, and/or a reduction insymptoms. The application of these pharmaceutical compositions canreduce S. aureus in the skin of the subject being treated. Theapplication of the pharmaceutical composition can provide enhancedbarrier function of the skin as measured by trans-epidermal water loss.

Atopic dermatitis occurs as flare-ups, and there can be periods ofremission. The topical application can reduce reoccurrences, so thatadditional incidents of atopic dermatitis are reduced in number,intensity, or frequency. The topical application can increase the timeof remission, such as the length of time between incidents. In someembodiments, an additional incident of atopic dermatitis will not occurfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks followingapplication. In an additional embodiment, an additional incident ofatopic dermatitis will not occur for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months following the topical application.

The method can include measuring the microbiota of the skin of thesubject. Specifically, diagnostic assays can be performed to determineif the bacterial taxa in the skin of a subject is altered followingtreatment. Thus, in some embodiments, it is determined if the bacterialphyla, bacterial classes, bacterial orders, bacterial families,bacterial genera and/or bacterial species are altered in the skin of asubject with atopic dermatitis. In one embodiment, it is determined ifthe amount of S. aureus is modified in the skin of the subject followingtreatment.

Such a method for identifying a microbiota in a sample can includeproviding a sample, such as a skin sample, and detecting at least onemicrobiota in the sample. One embodiment of the method can includepreparing a nucleic acid sample including a molecular indicator ofidentity from at least one microbiota present in the sample anddetecting the molecular indicator of identity. For example, the methodcan involve preparing at least one nucleic acid sample by preparing aDNA sample. The molecular indicator of identity can be a polymorphicpolynucleotide, such as an rRNA gene (for example, a 16S rRNA gene). Themolecular indicator of identity can be detected by determining thenucleotide sequence of the polymorphic polynucleotide, such as the 16SrRNA gene, or a portion or subsequence thereof. Additional embodimentsfor detecting the molecular indicator of identity can also include PCRwith selective primers, quantitative PCR with selective primers, DNA-DNAhybridization, RNA-DNA hybridization, in situ hybridization, andcombinations thereof. For example, the polymorphic polynucleotide can bedetected by hybridization to a specific probe. In such an example, thespecific probe hybridizes to a polymorphic target nucleic acid, such asa 16S rRNA gene. Optionally, the nucleic acid can be hybridized to atleast one array comprising a plurality of specific probes, e.g., aplurality of specific probes, each of which identifies a bacteria.Detecting the molecular indicator of identity can also be accomplishedusing protein probes (such as antibodies) that bind to polymorphictarget proteins, for example polymorphic target proteins that identifythe microbiota (see U.S. Pat. No. 9,173,910, incorporated herein byreference).

The relative abundance of one or more bacteria, such as S. aureus, canbe measured in a sample from a subject. As used herein, the term“relative abundance” refers to the commonality or rarity of an organismrelative to other organisms in a defined location or community. Forexample, the relative abundance can be determined by generally measuringthe presence of a particular organism compared to the total presence oforganisms in a sample.

The relative abundance of bacteria can be measured directly orindirectly. Direct measurements can include culture based methods.Indirect measurements can include comparing the prevalence of amolecular indicator of identity, such as ribosomal RNA (rRNA) genesequences, specific for an organism or group of organisms in relation tothe overall sample.

In one embodiment, the relative abundance of microbiota, such S. aureusand/or any type of Gram negative bacteria, within the skin an individualsubject may be calculated by measuring the ratio of one or more specificbacteria in a sample from an individual to obtain a microbiota profileof the subject. The relative abundance can be derived from the totalabundance of bacteria present in a sample. As used herein, the “totalabundance” refers generally to the total bacteria in a sample. Thus, a“microbiota profile” refers to a representation, such as a graph, of therelative abundance of one or more microbiota in a subject or sample ofskin from a subject.

Gram Negative Bacteria

The disclosed a therapeutically effective amount of a purified viablegram negative bacteria can be provided as components of a kit. Thepurified viable gram negative bacteria can be provided in a growthmedium, in a lyophilized form, or as frozen cells. Thus, the kit caninclude a container comprising a therapeutically effective amount of apurified viable gram negative bacteria, wherein i) a lysate and/orcomponent of the gram negative bacteria inhibits growth of S. aureus inan in vitro assay; ii) the gram negative bacteria stimulates humankeratinocytes iii) the gram negative bacteria induces cytokineexpression from human cells; and iv) the gram negative bacteria isnon-pathogenic when administered to the skin of the subject.

In some embodiments, the kit can include the components needed toproduce a pharmaceutical composition, such as one container includingthe gram negative bacteria (in any form) and one container including apharmaceutically acceptable carrier for suspending the gram negativebacteria thereof. The pharmaceutically acceptable carrier can be, forexample, a buffered saline solution or a sucrose solution. In otherembodiments, the kit can include a container including the gram negativebacteria, and a second container including a pharmaceutically acceptablecarrier, and a device, such as, but not limited to, a syringe, formeasuring the pharmaceutically acceptable carrier. In yet anotherembodiment, the kit can include a device, such as, but not limited to, aspray nozzle or a bandage, for topical application of the gram negativebacteria once it is suspended in the pharmaceutically acceptablecarrier.

Optionally, such a kit includes additional components includingpackaging, instructions and various other reagents, such as additionalbuffers or other therapeutic ingredients. The kit can include acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,tubes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container typically holds a compositionincluding the gram negative bacteria which is effective for treatingatopic dermatitis. In several embodiments the container may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The label or package insert indicates that thecomposition is used for treating the particular condition, such asatopic dermatitis.

The label or package insert typically will further include instructionsfor use. The package insert typically includes instructions customarilyincluded in commercial packages of therapeutic products that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts. In some non-limiting examples, the instructions includeinformation on the amount of the pharmaceutically acceptable carrier toadd to the vial containing the gram negative bacteria, instructions forsuspending the gram negative bacteria in the pharmaceutically acceptablecarrier, and instructions for topical application to the skin. Theapplication can be spraying on the skin, swabbing on the skin, orintroducing the suspension onto a bandage for application to the skinThe instructional materials may be written, in an electronic form (suchas a computer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed, such as spraytips, bandages, or swabs for dermal application. The kits mayadditionally include buffers and other reagents routinely used for thepractice of a particular method. Kits and appropriate contents are wellknown to those of skill in the art.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Studies were performed to evaluate if immunologic outcomes differ afterexposure to different commensal Gram-negative (CGN) bacteria collectedfrom human skin. For these studies, CGN bacteria were collected fromhealthy controls and patients with atopic dermatitis (AD). Using variouscellular and culture-based models, their immunogenicity was evaluated.Representative strains of CGN were selected, and their impact wasevaluated in the MC903 mouse model of AD. It was found that CGN bacteriataken from healthy human volunteers but not from patients with AD wereassociated with enhanced barrier function, innate immunity activation,and control of S. aureus. Treatment of AD with CGN from healthy controlsimproved outcomes in a mouse model.

Example 1 Materials and Methods

Gram-Negative Bacterial Collection and Identification:

Two FloqSwabs (Copan, Brescia, Italy) moistened in sterile phosphatebuffered saline (PBS; Corning Cellgro, Corning, N.Y.) were rubbed on thesubject's skin (at the antecubital fossa) vigorously for 15-30 seconds.One swab was placed into a 15 mL conical tube (Corning Life, Corning,N.Y.) with 2 mL of sterile Hank's balanced salt solution (HBSS;Sigma-Aldrich) containing vancomycin (300 ug/mL) and amphotericin B (5ug/mL; Sigma-Aldrich, St. Louis, Mo.) to inhibit growth of Gram-positivebacteria and fungi. The remaining swab was placed into a 15 mL conicaltube containing 2 mL of R2A (Reasoner's 2A) broth (Teknova, Hollister,Calif.) with similar concentrations of vancomycin and amphotericin B.The tubes, with swabs left in place, were then incubated at 32° C. withconstant shaking for 48-72 hours before plating 100 uL from each tubeonto an R2A agar plate (Remel, Lenexa, Kans.). Colonies were then takenfor species identification by mass spectrometry using matrix-assistedlaser desorption/ionization-time of flight (MALDI-TOF) analysis.Bacterial protein extraction for MALDI-TOF MS using the BioTyper (v3.1,Bruker Daltonics Inc., Billerica, Mass.) was performed by the NIHClinical Center microbiology lab using previously described methods (Lauet al., Journal of clinical microbiology 52, 2804-2812 (2014); publishedonline Epub August (10.1128/JCM.00694-14), instrument settings andcalibration (Lau et al., Journal of clinical microbiology 51, 828-834(2013); published online (Epub) March (10.1128/JCM.02852-12); Youn etal., Journal of clinical microbiology, (2015); published online (Epub)September 2 (10.1128/JCM.01643-15)). BioTyper identification wassupplemented by additional mass spectra profiles provided by several NIHdeveloped databases (Lau et al, 2014, supra; Stevenson et al., Journalof clinical microbiology 48, 3482-3486 (2010); published online (Epub)October (10.1128/JCM.00687-09; Myles et al., Nature immunology 14,804-811 (2013); published online (Epub) August (10.1038/ni.2637)). Tenof the healthy control R. mucosa strains reported in FIG. 1 wereidentified based solely on the unique colony morphology, UV lightreactivity, and Gram-stain characteristics that had been observed in theother R. mucosa isolates identified by MALDI-TOF analysis. All R. mucosaisolates used for subsequent studies were verified by MALDI-TOFanalysis. Written informed consent was obtained for all participants inthis study.

In vitro Staphylococcus aureus inhibition assay: Gram-negative isolateswere cultured as above in 5 mL of R2A for 8-10 days. Bacteria werepelleted at 5000 g for 12 minutes. Supernatants were harvested, frozen,then lyophilized (Labconco FreeZone 2.5, Kansas City, Mo.). Thelyophilized product, and a control of concurrently incubated andlyophilized R2A without bacteria, were suspended in 1 mL of tryptic soybroth (TSB). An overnight culture of S. aureus strains grown in 5 mL TSBwas diluted 1:100 into fresh media then 100 mcL of the newly dilutedculture was combined with 100 mcL of the TSB containing the CGNsupernatant or lyophilized R2A control. Samples were incubated for 3hours at 37° C. under constant agitation. Serial dilutions wereperformed and plated; the following day colonies were counted andaveraged across all countable plates. Percent impact on S. aureus growthwas calculated by dividing the number of CFU in the CGN-supernatantexposed culture by the number of CFU in the R2A-only exposed culture.

Blister Induction and Bacterial Challenge:

An eight-well suction chamber was designed (Ammnra Creations; San Jose,Calif.) and 3D printed in bronze (Shapeways; New York, N.Y.) (FIG. S1A).This was placed onto the participants' ethanol-sterilized volar forearm,and placed under 30 mm Hg of suction for 2 hours using amicrodermabrasion device (Kendal Diamond HB-SF02). After device removal,the resultant epidermal blister roofs were surgically removed. Eightchallenge chambers were designed (Ammnra Creations) and 3D printed inbronze (Shapeways) (FIG. 4C). Using a matching eight well template(Ammnra Creations), 10 mm punch biopsies (Acu-Punch, Acu-derm; FortLauderdale, Fla.) were cut into one 4″×4″ Duoderm dressing (ConvaTec;Bridgewater, N.J.). The rims of the challenge chambers were brushed withDermabond adhesive (Ethicon; Somerville, N.J.) before being placed overthe denuded blisters. Each well was filled with 1 mL of either sterilesaline (Corning Life) or 2e7 CFU of irradiated bacteria suspended insterile saline. Blister fluid was removed via pipette (Eppendorf;Hauppauge, N.Y.) the following morning. Collected samples werecentrifuged at 350 g for 7 minutes, supernatants were removed and frozenfor batch analysis.

Cytokine and Anti-Microbial Peptide Detection:

Standards of human reg3gamma (Sino Biologicals; Beijing, China) orblister fluid samples (100-12.5% diluted in 0.9% NaCl) were coated onNunc Maxisorp plates overnight. The next day, wells were washed 5× withPBS and blocked in 3% BSA in PBS for 1 h. Wells were washed once withPBS. Polyclonal, protein A-purified rabbit anti-mouse reg3gamma (13.2mg/ml; kind gift from J. Kolls, University of Pittsburgh) was added at a1:1000 dilution in 1% BSA/PBS and incubated 90 min at RT. Anti-rabbitHRP (Santa Cruz; Dallas, Tex.) and TMB substrate (Ebioscience; SanDiego, Calif.) were added and plate was incubated at RT for 5-10 min. 2NH₂SO₄ was added to stop the reaction; plates were read at 450 nm. LL-37(Hycult; Plymouth Meeting, Pa.) and human beta-defensin 3 (PeproTech;Rocky Hill, N.J.) were measured by commercial ELISA kits. Cytokinelevels were determined by Bio-Plex (BIO-Rad; Hercules, Calif.) permanufacturer instructions.

Keratinocyte Cultures:

Primary foreskin keratinocyte (KC) cultures were collected andstimulated as previously described (Myles et al., Nature immunology 14,804-811 (2013); published online EpubAug (10.1038/ni.2637)). 1e7 CFU oflive commensal Gram-negative bacteria were added to KC culture media.mRNA was extracted after 24 hours for PCR analysis by the ΔΔCT method.

Mice:

BALB/c mice were purchased from Taconic Farms (Hudson, N.Y.). Mice wereused between 8 and 14 weeks of age. Experiments were performed in bothmale and female mice, but age and sex matched within each experiment.

Trans-Epidermal Water Loss (TEWL) Measurements:

Mice were shaved and hair chemically removed (Nair). Starting thefollowing day, approximately 1e7 CFU of live bacteria were placed on thenude areas daily. Immediately prior to inoculation, TEWL was measureddaily by VapoMeter (Delfin; Greenwich, Conn.), per manufacturer'sinstructions.

MC903 and Ear Inoculations:

MC903 mouse model of AD was performed as previously described (Wang etal., The Journal of allergy and clinical immunology 135, 781-791 e783(2015); published online (Epub) March (10.1016/j.jaci.2014.09.015)). Forprevention studies, 1e7 CFU of Gram-negative bacteria were suspended insterile PBS and dripped onto the mouse ears in 10 mcL of volume.Inoculations were initiated two days prior to MC903, and continuedthroughout the MC903 exposure. MC903 was placed first, the ethanol wasallowed to evaporate for 2-5 minutes prior to placement of bacterialisolates. Ear thickness was measured on day 14. mRNA isolation and PCRwere performed as previously described (Myles et al., 2013, supra). Forexperiments using co-inoculation of S. aureus, 1e6 CFU of the SAAS9strain of S. aureus was dripped onto the ear immediately prior toinoculation with the Gram-negative isolate. Treatment studies wereperformed by exposing mice to MC903 daily for 14 days and inoculatingwith 1e7 CFU of Gram-negative bacteria on days 13-15. Ear thickness wasmeasured and photos taken on day 21.

Serum Total IgE Analysis:

Serum was collected on day 14 of MC903 treatment. Total IgE wasdetermined as previously described (Myles, Nutrition journal 13, 61(2014)10.1186/1475-2891-13-61)) using a commercial kit (BethylLaboratories, Montgomery, Tex.).

Statistics:

Means were compared using two-tailed unpaired t test, or ANOVA forcomparison of multiple samples, with Prism software (GraphPad, SanDiego, Calif.). NS=not significant, *=p<0.05, **=p<0.01, ***=p<0.001,****=p<0.0001.

Study Approval:

All animal experiments were conducted under approved Office of AnimalCare and Use procedures. All human sample collection and processing wasperformed under IRB approved clinical trials and all subjects gave fullconsent to sample collection. All participants provided their writtenconsent to the research protocol and institutional review board (IRB)consent was obtained prior to blood collection.

Example 2 Skin Commensal Gram-Negative Microbiota Differ Between HealthyControls and Patients with AD

Genetic-based microbiome identification has revealed significantdifferences in the Gram-negative skin biome between AD patients andhealthy controls (4). A method for culturing Gram-negative skin florathrough appropriate selection of media, antibiotics and temperatureallowed for functional characterization and comparison of CGN species.Significant differences were found in the culturable bacteria present onthe skin of 17 AD patients as compared to 26 healthy volunteers (FIG.1A; Table 1).

TABLE 1 Demographics of controls and patients. Demographic ControlsPatients Significance Number 26 17 — Age Mean (range) 32.5 (8-65) 18.5(8-51) ** Sex (%) Male 50 70 NS Female 50 30 Race (%) White 39 47 NSBlack 15 18 Latino  0  0 Asian 31 24 Other/Mixed 15 11 SCORAD (range) 0(0) 19.7 (1-56) **** Age, sex, race, and SCORAD for participantsincluded in FIG. 1. Significance determined by Student's t test (age,SCORAD) or Chi squared (sex, race). ** = p < 0.01, **** = p < 0.001, NS= not significant.

The predominant Gram-negative species identified in healthy volunteers(HV) was Roseomonas mucosa. Roughly half of AD patients did not have anyculturable Gram-negative bacteria, consistent with DNA-based analysis(Kong et al., Genome research 22, 850-859 (2012); published onlineEpubMay (10.1101/gr.131029.111)).

Example 3 CGN from Healthy Volunteers Inhibit the Growth of S. aureus

Overgrowth and infection with S. aureus is both a contributor to, andconsequence of the immune imbalance and poor barrier functioncharacteristic of AD. S. aureus can directly activate allergic mastcells (Schlievert et al., J Allergy Clin Immunol 125, 39-49 (2010);published online (EpubJan) (10.1016/j.jaci.2009.10.039; Nakamura et al.,Nature 503, 397-401 (2013); published online (Epub) November 21(10.1038/nature12655)) and T-cells (Brauweiler et al., The Journal ofinvestigative dermatology 134, 2114-2121 (2014); published online (Epub)August (10.1038/jid.2014.43)). Treatment with antibiotics can reduce S.aureus burdens and improve symptoms, but do not normalize the underlyingpathology (Boguniewicz and Leung, J Allergy Clin Immunol 132, 511-512e515 (2013); published online (Epub) August(10.1016/j.jaci.2013.06.030)). To evaluate the impact of the CGN strainson S. aureus growth, multiple isolates of S. aureus were grown in thepresence of the supernatant from cultures of CGN. On average,supernatants from HV-CGN inhibited S. aureus by nearly 50% (FIG. 1B;FIG. 4). In contrast, AD-CGN had more variable effects, with moststrains failing to inhibit (FIG. 1B; FIG. 4). Re-inoculation of S.aureus from the inhibitory CGN supernatants into fresh media allowednormal growth, suggesting bacteriostatic rather than bactericidalactivity. Consistent with this in vitro analysis, co-inoculation of CGNand S. aureus on mouse ears also reduced S. aureus yields (FIG. 1C).This inhibition was associated with production of the lipidlysophosphatidylcholine (LPC) and recapitulated using commerciallypurchased LPC (FIG. 8B) indicating unique features of our bacteria wereassociated with potential clinical benefit.

Example 4 CGN from Healthy Volunteers Induce Innate Immunity in Humans

To measure in vivo human cutaneous immune reactivity to these bacteria,suction blisters were induced on the forearms of healthy volunteers(FIG. 5A) and the epidermal blister roof was removed (FIG. 5B) similarlyto what has been previously described (Follin and Dahlgren, Methods inmolecular biology 412, 333-346 (2007)10.1007/978-1-59745-467-4_22).Challenge chambers (FIG. 5C) were used to expose the dermal blister baseto lethally irradiated isolates of either an HV-derived or AD-derived R.mucosa, chosen based on their disparate inhibition of S. aureus (FIG.1B).

After 20-24 hours of stimulation, blister cytokines levels for IL-6(FIG. 2A-2B) were significantly higher in response to the HV R. mucosacompared to the one from a patient with AD (FIG. 2A-2B). There were nodifferences in levels of traditional Th (T-helper) cytokines such asinterleukin (IL)-17, interferon gamma (IFNγ), or IL-4 (FIG. 2A), norwere there significant differences in levels of many other cytokines(FIG. 6A) or antimicrobial peptides (FIG. 6B); however, adaptive T-cellcytokines should also be measured at later time points. Infection ofhuman foreskin-derived primary keratinocytes (KC) with multiple isolatesof live CGN in vitro showed variable effects of the different isolateson KC innate immunity markers, but compared to AD-CGN, HV-CGN enhancedmRNA abundance of defensin β4A (FIG. 7A), and the upstream modulators(Miller et al., Dermatologic therapy 23, 13-22 (2010); published online(Epub) January-February (10.1111/j. 1529-8019.2009.01287.x); Schauberand Gallo, Experimental dermatology 17, 633-639 (2008); published online(Epub) August (10.11111/j.1600-0625.2008.00768.x).) CYP27b1 (a vitamin Dconverting enzyme; FIG. 7B) the vitamin D receptor (VDR) (FIG. 7C), andthe anti-microbial peptide cathelicidin (FIG. 7D). There were nodifferences in transcript levels for IL-10 or IL-6 (FIG. 7E-7F). CD14,IL-8, Tumor Necrosis Factor alpha (TNFα, Toll Like Receptor (TLR) 2,TLR3, TLR4, TLR9, or Thymic stromal lymphopoietin (TSLP) mRNA abundancewas also not different, and there was no apparent correlation betweenthe ability of an isolate to inhibit S. aureus and activate KC.

Example 5 CGN from Healthy Volunteers Preserves Barrier Function in Mice

The loss of barrier function in AD causes dry, itchy skin due totrans-epidermal water loss (TEWL) (Boguniewicz et al., J Allergy ClinImmunol 125, 4-13; quiz 14-15 (2010); published online (Epub) January(10.1016/j.jaci.2009.11.027)) and cutaneous sensitization to antigens(Pyun, Allergy, asthma & immunology research 7, 101-105 (2015);published online (Epub) March (10.4168/aair.2015.7.2.101)). For a subsetof patients, this barrier defect is associated with dysfunction in thetight-junction protein filaggrin (Bantz et al., Journal of clinical &cellular immunology 5, (2014); published online (Epub) April(10.4172/2155-9899.1000202)). Topical application to healthy mouse earsof the representative isolate of HV-CGN used in the human blisterstudies enhanced transcript levels of filaggrin compared to the AD-CGNisolate (FIG. 2C) without any noticeable change in ear erythema orthickness. Application of AD-CGN increased TEWL, while HV-CGN had noeffect (FIG. 2D). Taken together, these data suggest that strains of CGNassociated with a healthy status induce potentially beneficial immuneoutcomes related to activation of vitamin D, stimulation of innateimmunity, and preservation of barrier function.

Example 6 CGN from Healthy Volunteers Improve Outcomes in a Mouse Modelof AD

MC903, a vitamin D analogue, induces an AD-like dermatitis when appliedto mouse ears (22). Concurrent application of the HV-sourced R. mucosaisolate protected against onset of MC903-induced dermatitis as measuredby ear thickness (FIG. 3A). In contrast, the AD-sourced R. mucosaisolate failed to protect against disease onset as did the HV-sourced P.aeruginosa (FIG. 3A), despite the latter's impact on KC activation (FIG.7) and S. aureus inhibition (FIG. 1B). Application of the AD-sourced R.mucosa enhanced serum IgE induction, whereas the HV-sourced R. mucosashowed no significant impact (FIG. 3B). Consistent with inoculation ofhealthy mouse ears, transcript levels of filaggrin were significantlylower in MC903-treated mice exposed to AD-sourced R. mucosa (FIG. 9).

To test the therapeutic potential of CGN, AD-like dermatitis was inducedwith MC903 and then the ears were inoculated with CGN daily for threedays. Treatment with HV-R. mucosa was associated with a reduction in earthickness and visible redness (FIG. 3C-3D). To evaluate if a livebiotherapeutic was necessary, or if surface and secreted factors alonecould provide similar results, we applied an equivalent CFUconcentration of lethally irradiated HV-CGN suspended in itssupernatant. Neither this ‘dead mix’ nor the supernatant alone providedany benefit, and each increased ear thickness (FIG. 3C-3D).

While genome-wide linkage studies (Barnes et al., J Allergy Clin Immunol125, 16-29 e11-11; quiz 30-11 (2010); published online (Epub) January(10.1016/j.jaci.2009.11.008)) and S. aureus characterization haveelucidated the pathogenesis of AD, many causal underpinnings remainenigmatic. Current therapeutic approaches targeting host response and S.aureus colonization can significantly improve AD symptoms, yet even whensuccessful, these treatments are not curative and extract a significantemotional toll on patients and their families (24). The studiesdisclosed herein evidence that the reported dysbiosis in AD is notsimply an associated finding, but can be a contributor to pathology.Strains of R. mucosa isolated from healthy controls were able toinfluence many hallmarks of AD, improving barrier function, enhancinginnate activation, and limiting S. aureus growth. Other Gram negativebacteria could provide a similar effect.

Addition of Gram-negative V. filiformis lysates to skin creams mayprovide benefit in AD treatment (Gueniche et al., The British journal ofdermatology 159, 1357-1363 (2008); published online (Epub) December(10.1111/j.1365-2133.2008.08836.x). However, the lack of efficacy ofkilled R. mucosa suggests that use of isolated secreted and/or surfaceproducts without live commensals may not provide benefit. Without beingbound by theory, the dynamic interaction of host-commensal may berequired for clinical utility of R. mucosa, and the potential forcolonization offers the advantages of limited reapplications and morephysiologic pharmacokinetics.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A pharmaceutical composition, wherein the pharmaceuticalcomposition comprises: at least one strain of Roseomonas mucosa, whereinthe at least one strain of Roseomonas mucosa is a viable, skin commensalorganism present in an amount sufficient for reducing growth ofStaphylococcus aureus on skin of a subject in need thereof, and whereinthe pharmaceutical composition is formulated in a topical dosage formselected from the group consisting of a cream, gel, foam, ointment, anda lotion.
 2. The pharmaceutical composition of claim 1, wherein the atleast one strain of Roseomonas mucosa comprises at least two strains ofRoseomonas mucosa.
 3. The pharmaceutical composition of claim 1, whereinthe at least one strain of Roseomonas mucosa comprises three strains ofRoseomonas mucosa.
 4. The pharmaceutical composition of claim 1, furthercomprising a pharmaceutically acceptable carrier.
 5. The pharmaceuticalcomposition of claim 4, wherein the pharmaceutically acceptable carriercomprises sucrose.
 6. The pharmaceutical composition of claim 1, whereinthe at least one strain of Roseomonas mucosa is purified.
 7. Thepharmaceutical composition of claim 1, wherein the at least one strainof Roseomonas mucosa is present in an amount of 10⁴ to 10¹² colonyforming units.
 8. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is contained within a spray bottle.